Phenylalanine derivatives for treatment of measles infections

ABSTRACT

Pharmaceutical compositions in dosage unit form comprising a pharmaceutical carrier and N-carbobenzoxy-D-phenylalanyl-Dphenylalanine or a pharmaceutically-acceptable salt. Methods for the prophylaxis or treatment of measles infections, by administering N-carbobenzoxy-D-phenylalanyl-D-phenylalanine or a pharmaceutically-acceptable salt.

Unite States Patent [151 3,700,770

Nicolaides 1 Oct. 24, 1972 [54] PHENYLALANINE DERIVATIVES FOR [56] References Cited TREATMENT OF MEASLES INFECTIONS Ernest D. Nicolaides, Ann Arbor, Mich.

Parke, Davis & Company, Detroit, Mich.

Filed: 0121.21, 1966 Appl. No.: 588,306

Inventor:

Assignee:

US. Cl ..424/ 177 Int. Cl. ..A61k 27/00 Field of Search 167/652; 424/177 OTHER PUBLICATIONS Chemical Abstracts 46: 6103 h (1952) Chemical Abstracts 47: 6378 b (1953) Primary ExaminerJerome D. Goldberg Attomey-Robert R. Adams, David B. Ehrlinger, George M. Richards and Edward J. Gall ABSTRACT 5 Claims, No Drawings PHENYLALANINE DERIVATIVES FOR TREATMENT OF MEASLES INFECTIONS The present invention relates to amino acid compounds. More particularly, the invention relates to N- carbonbenzoxy-D-phenylalanyl-D-phenylalanine of the formula .942. arr-.1

to pharmaceutically-acceptable salts thereof, to pharmaceutical compositions containing the foregoing compounds, and to methods for the use of the foregoing compounds as anti-measles viral agents.

In the description of this invention, the terms D- phenylalanyl and D-phenylalanine are used in accordance with presently accepted amino acid terminology to indicate that the structures belong to the same configurational series as D-(dextro)-g1yceraldehyde and thus are opposite in configuration to the amino acids of the so-called natural series which are most commonly isolated from proteins and other biological sources.

The term "pharmaceutically-acceptable salt as used herein includes any salt which is not substantially more toxic than an equal weight of the N-carbobenzoxy-D- phenylalanyl-D-phenylalanine from which it is derived when measured in the same mammalian host using the same vehicle and method of administration. Some examples of such salts are the sodium, potassium, calcium, ammonium, choline, 2-hydroxyethylammonium, and other salts formed with various organic and inorganic bases. The preferred salts are the pharmaceutically-acceptable salts with an alkali metal, an alkaline earth metal, ammonia, or an amine.

In accordance with the invention, pharmaceutical compositions are produced by formulating N-carbobenzoxy-D-phenylalanyl-D-phenylalanine or a pharmaceutically-acceptable salt thereof in dosage unit form with a pharmaceutical carrier. Some examples of dosage unit forms are tablets, capsules, lozenges, and pills; as well as powders and aqueous and non-aqueous solutions and suspensions packaged in containers containing either one or some larger number of dosage units and capable of being subdivided into individual doses by such means as measurement into a teaspoon or other standard container. Some examples of suitable pharmaceutical carriers, including pharmaceutical diluents, are gelatin capsules; sugars such as lactose and sucrose, starches such as corn starch and potato starch; cellulose derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, and cellulose acetate phthalate; gelatin; talc; stearic acid; magnesium stearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil of theobroma; propylene glycol; glycerine; sorbitol; polyethylene glycol; water; agar; alginic acid; isotonic saline; and phosphate buffer solutions; as well as other compatible substances normally used in pharmaceutical formulations. The compositions of the invention can also contain other components such as coloring agents, flavoring agents, and/or preservatives. These materials, if present, are usually used in relatively small amounts. The compositions can, if desired, also contain other therapeutic agents.

The percentage of the N-carbobenzoxy-D-phenylalanyl-D-phenylalanine or a pharmaceutically-acceptable salt thereof in the foregoing compositions can be varied within wide limits but for practical purposes it is preferably present in a concentration of at least 5 percent. The most satisfactory compositions are those in which a much higher proportion of the N-carbobenzoxy-D-phenylalanyl-D-phenylalanine or a pharmaceutically-acceptable salt thereof is present. The compositions of the invention preferably contain from 25 to 1,000 mg. of the indicated active ingredient per dosage unit so that the entire amount to be administered during a day can be made up from a reasonable number of dosage units.

Also in accordance with the invention, N-carbobenzoxy-D-phenylalanyl-D-phenylalanine and pharmaceutically-acceptable saltsthereof are employed as anti-measles viral agents. For this purpose they are administered in dosage unit form for the prophylaxis and treatment of measles viral diseases. The aforementioned compounds and compositions containing the same can be administered either orally or parenterally, with the dose adjusted to the needs and tolerances of the individual patients. A specific use of these substances for their anti-measles viral activity is as an adjunct to measles vaccine treatment. Thus, a person is vaccinated with an attenuated measles vaccine; and 2 to 4 days following vaccination, N-carbobenzoxy-D- phenylalanyl-D-phenylalanine or a pharmaceuticallyacceptable salt thereof is administered. Administration of the drug after this lapse of time minimizes the fever and other side effects sometimes caused by attenuated measles vaccine without unduly retarding the formation of antibodies. N-carbobenzoxy-D-phenylalanyl-D- phenylalanine and its pharmaceutically-acceptable salts can also be used prophylactically to prevent a measles infection in an exposed person and therapeutically in the treatment of an established measles infection. For parenteral administration, a suitable total daily dose would amount to mg. or more ranging up to a limit of 15 g. in exceptional cases. For oral administration, a suitable total daily dose would amount to 250 mg. or more ranging up to a limit of 30 g. in exceptional cases. The exceptional cases indicated would be adults tolerating the substances well, in whom the maximum possible effect is desired. In general, treatment is continued for a period of no longer than a few days.

Further in accordance with the invention, N-carbobenzoxy-D-phenylalanyl-D-phenylalanine and its salts are provided in purified crystalline forms with well-defined physical characteristics. A compound previously reported as N-carbobenzoxy-D-phenylalanyl-D-phenylalanine in free acid form was described as an oily material; see Chemical Abstracts, 46, 6l03- 6104 (1952). By the present invention, N-carbobenzoxy-D-phenylalanyl-D-phenylalanine in free acid form is provided as a purified crystalline material, m.p. substantially 156l57 C. subject to the usual variations encountered in experimental determina- 3 tions, and having other physical properties as described in the examples to follow.

Purified crystalline N-carbobenzoxy-D-phenylalanyl- D-phenylalanine can be prepared, according to the invention, as follows. D-phenylalanine is reacted with methanol in the presence of a mineral acid to produce D-phenylalanine methyl ester hydrochloride. N-carbobenzoxy-D-phenylalanine is reacted with ethyl chloroformate in the presence of triethylamine to produce the anhydride of N-carbobenzoxy-D-phenylalanine with ethyl hydrogen carbonate, and the latter compound is reacted with D-phenylalanine methyl ester hydrochloride in the presence of triethylamine to produce N-carbobenzoxy-D-phenylalanyl-D-phenylalanine methyl ester. The latter compound is reacted with sodium hydroxide in an aqueous alkanol, and the product collected following acidification and then crystallized from ethyl acetate-petroleum ether to afford purified crystalline N-carbobenzoxy-D-phenylalanyl-D-phenylalanine. Alternatively, according to the invention, N-carbobenzoxy-D-phenylalanine and p- 'nitrophenol are reacted in the presence of N,N-

dicyclohexylcarbodiimide to produce N-carbobenzoxy- D-phenylalanine p-nitrophenyl ester. This compound is reacted with D-phenylalanine methyl ester (obtained by basifying the hydrochloride with triethylamine) to produce N-carbobenzoxy-D-phenylalanyl-D-phenylalanine methyl ester. The latter compound is reacted with sodium hydroxide in an aqueous alkanol, and the product collected following acidification and then crystallized from acetone-petroleum ether to afford purified crystalline N-carbobenzoxy-D-phenylalanyl-D- phenylalanine.

. Purified crystalline salts of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine are also obtained according to the present invention. Such salts are formed by reaction of Ncarbobenzoxy-D-phenylalanyl-D-phenylalanine with any of a variety of organic and inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, ammonia and amines. Such salts differ in solubility properties from N-carbobenzoxy-D-phenylaIanyLD-phenylalanine but are otherwise equivalent for the purposes of the invention.

The compounds, compositions, and methods of the present invention are useful in anti-measles viral therapy. They can be employed in the prophylaxis and treatment of measles. They are also useful following the administration of an attenuated measles vaccine as a means of minimizing the fever and other side effects sometimes caused by such vaccines.

The anti-measles viral activity of N-carbobenzoxy-D- phenylalanyl-D-phenylalanine and its pharmaceutically-acceptable salts can be measured quantitatively in tissue culture systems. For example, tissue culture systems are prepared from H.Ep.No.2 cells (an established cell line) and inoculated with measles Various concentrations of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine are added and the tissue cultures are observed over a period of several days. Antiviral activity is demonstrated by the absence of the cytopathogenic effect normally caused by the virus. Alternatively, an experimental animal such as a rat or a dog is given N-carbobenzoxy-D-phenylalanyl-D-phenylalanine by intraperitoneal injection and at various intervals beginning 1 hour later the animals are bled. Samples of the blood sera are added to the H.Ep.No.2 tissue cultures inoculated with measles virus and the anti-measles viral activity of the sera determined by the absence of the cytopathogenic effect.

The invention is illustrated by the following examples.

EXAMPLE 1 A mixture of 50 g. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine, 60 g. of powered milk sugar, g. of powdered sugar containing 3 percent starch, and 15 g. of sifted potato starch is blended in a twin-shell blender. The resulting powder mixture is granulated with a 20 percent solution of acacia in water containing alcohol as a preservative. A quantity of acacia solution equivalent to 1.8 g. of dry acacia is used and the granulation is finished with water if necessary. The wet granulation is screened through a No. 10 mesh stainless steel screen and dried overnight in a hot air drier at 50-55 C. The dried granulation ispassed through. a No. 20 mesh screen and blended with 8 g. of sifted talc, 10 g. of sifted potato starch, and 1.8 g. of magnesium stearate. The mixture is processed through a rotary tableting machine using inch concave, single breakline punches. Yield equals approximately 1,000 tablets, each containing 50 mg. of N-carbobenzoxy-D- phenylalanyl-D-phenylalanine.

EXAMPLE 2 A mixture of 50 g. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine and 170 g. of powdered milk sugar is passed through a No. 30 mesh screen and blended thoroughly in a twin-shell blender. The powdered mixture is filled into No. 3 opaque, hard gelatin capsules. Yield equals approximately 1,000 capsules, each con taining 50 mg. of N-carbobenzoxy-D-phenylalanyl-D- phenylalanine.

EXAMPLE 3 EXAMPLE 4 A solution is prepared by dissolving 0.902 g. of sodium hydroxide in 400 cc. of distilled water. With stirring, 10 g. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine and cc. of propylene glycol are added. Stirring is continued until a clear solution is obtained and the 200 g. of cane sugar, medium granular and 2 mg. of sodium saccharin are added and dissolved. A mixture of 0.60 cc. of raspberry flavor and 4 cc. of alcohol is added and the solution is adjusted with hydrochloric acid to pH 7.2 and diluted to 1,000 cc. with distilled water. The solution is filtered and filled into 4 ounce bottles. Each 5 cc. dose contains 50 mg. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine.

EXAMPLE 5 A mixture is prepared by stirring 3 g. of a gel-forming complex magnesium-aluminum silicate in 400 cc. of water. A product such as Veegum, HV can be used. One hundred cc. of glycerin and g. of N-carbobenzoxy-D-phenylalanyl-Dphenylalanine are added followed by 2 g. of sodium carboxymethyl cellulose. After the latter component has dispersed, 100 g. of cane sugar, medium granular and 2 mg. of sodium saccharin are added followed by a solution of 0.6 cc. of raspberry flavor in 4 cc. of alcohol. The mixture is adjusted to pH 4.5 with hydrochloric acid, diluted to 1,000 cc. with distilled water, homogenized and filled into 4 ounce bottles. The resulting suspension for oral administration contains 50 mg. of N-carbobenzoxy-D- phenylalanyl-D-phenylalanine per 5 cc. dose.

EXAMPLE 6 N-carbobenzoxy-D-phenylalanyl-D-phenylalanine, 50 g., is gas sterilized with ethylene oxide, aseptically dissolved in 250 cc. of sterile alcohol and precipitated in 650 cc. of sterile distilled water containing 1.5 g. of polyvinylpyrrolidone. The precipitated product is aseptically filtered, rinsed with sterile distilled water, and added while still moist to a vehicle prepared from 1.5 g. of polyvinylpyrrolidone and 0.4 g. of polyoxyethylene sorbitan monostearate in 1,000 cc. of sterile distilled water. A product such as polysorbate 60 or Tween 60 can be used. The mixture is passed through a homogenizer and filled into 2 cc. ampoules. The resulting suspension for parenteral administration contains 100 mg. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine per ampoule or 50 mg. per cc.

EXAMPLE 7 A paste is prepared by stirring 50 g. of N-carbobenzoxy-Dphenylalanyl-D-phenylalanine with 300 cc. of sterile distilled water. Sodium hydroxide, 4.52 g., is added followed by 600 cc. of polyethylene glycol 400. The mixture is heated to 65 C. until a clear solution is obtained. It is then allowed to cool to room temperature and is adjusted to pH 7.6 with dilute hydrochloric acid, iron-free reagent grade. The mixture is diluted to 1,000 cc. with sterile distilled water, filtered to clarify the solution and then filtered through a millipore (0.45 micron membrane. It is then filled into 2 cc. ampoules. The resulting solution for parenteral administration contains 100 mg. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine per ampoule or 50 mg. per cc.

EXAMPLE 8 Crystalline N-carbobenzoxy-D-phenylalanyl-D- phenylalanine is prepared as follows.

Dry hydrogen chloride is passed at a rapid rate over a period of 10 minutes into a suspension of 50 g. of D- phenylalanine in 400 ml. of methanol. The resulting solution is heated at reflux for 2 hours and then evaporated under reduced pressure. The residue is dis solved in 200 ml. of methanol and the solution is again evaporated under reduced pressure. The residue is dissolved in 400 ml. of methanol and dry hydrogen chloride is passed into the solution at a rapid rate over 21 period of 10 minutes. The solution is heated at reflux for 2 hours and evaporated under reduced pressure. The residue is dissolved in 200 ml. of methanol and the solution is again evaporated under reduced pressure. The dissolution in methanol and evaporation is repeated and the residue is dissolved in 100 ml. of methanol. The methanol solution is diluted with dry ether to crystallize the product, D-phenylalanine methyl ester hydrochloride; m.p. l59161 C.; [04 =18.5 (2 percent in methanol).

With stirring at 10 C., 3.4 g. of triethylamine and then 3.7 g. ethyl chloroformate are added to a solution of 10 g. of N-carbobenzoxy-D-phenylalanine in 150 ml. of methylene chloride. The resulting solution containing the anhydride of N-carbobenzoxy-D-phenylalanine with ethyl hydrogen carbonate is stirred at l0 C. for 20 minutes and a mixture of 7.2 g. of D-phenylalanine methyl ester hydrochloride and 3.4 g. of triethylamine in 50 ml. of methylene chloride is added. The solution is stirred for 2 hours at 0 C. and then 16 hours at 20-25 C. The solution is washed successively with ml. of water, 75 ml. of 5 percent aqueous sodium bicarbonate, 75 ml. of water, 75 ml. of 1N hydrochloric acid and 75 ml. of water, then dried and evaporated to a volume of about 50 ml. The solution is diluted slowly with petroleum ether to crystallize the product, N-carbobenzoxy-D-phenylalanylD-phenylalanine methyl ester; m.p. 145146 C. after recrystallization from ethyl acetate-petroleum ether; [111 204 (2 percent in dimethylformamide).

A solution is prepared by dissolving 8.0 g. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine methyl ester in ml. of methanol and is treated with 10 ml. of 2N aqueous sodium hydroxide. The solution is stirred for 90 minutes at 2025 C. and then treated with 115 ml. of water followed by 11 m1. of 2N hydrochloric acid. The insoluble product is collected on a filter, washed with water, dried and crystallized from ethyl acetate-petroleum ether. It is N-carbobenzoxy-D-phenylalanyl-D-phenylalanine; m.p. 156-l57 C.; [0:1 P 133 (2 percent in dimethylformamide).

EXAMPLE 9 Crystalline N-carbobenzoxy-D-phenylalanylD- phenylalanine is also prepared as follows.

A solution is prepared by dissolving 1.99 kg. of N- carbobenzoxy-D-phenylalanine and 1.02 kg. of pnitrophenol in 13.3 liters of ethyl acetate. The solution is chilled to 0-5 C. With stirring and external cooling to maintain the temperature at 513 C., a cold solution of 1.41 kg. of N,N'-dicyclohexylcarbodiimide in 3.2 liters of ethyl acetate is added gradually over a period of 15 minutes. The reaction mixture is then stirred at 010 C. for 2 hours and at 25 C. for 1 hour. The insoluble by-product, N,N'-dicyclohexylurea, is removed by filtration and the ethyl acetate filtrate containing N-carbobenzoxy-D-phenylalanine p-nitrophenyl ester is held at 5 C. until it is used.

With stirring, 682 g. of triethylamine and then 5.3 liters of ethyl acetate are added to a solution of 1.44 kg. of D-phenylalanine methyl ester hydrochloride in 2.0 liters of dirnethylforrnarnide. The reaction mixture is stirred at 25 C. for 15 minutes and the insoluble byproduct, triethylamine hydrochloride, is removed by filtration. The filtrate contains D-phenylalanine methyl ester (free base).

The ethyl acetate solution of N-carbobenzoxy-D- phenylalanine p-nitrophenyl ester, prepared as above, and the ethyl acetate solution of D-phenylalanine methyl ester, also prepared as above, are combined and stirred for 3 days at 2025 C. The resulting solution is washed successively with 1N hydrochloric acid, water, 1M aqueous sodium carbonate, and saturated aqueous sodium chloride. The organic phase is stirred with activated charcoal and diatomaceous earth and filtered. (Products such as Darco 6-60 and Celite can be used). The filtrate is evaporated at reduced pressure to a volume of 14 liters and diluted with 28 liters of petroleum ether. The insoluble product is collected on a filter, washed with petroleum ether and dried. It is N-carbobenzoxy-D-phenylalanyl-D-phenylalanine methyl ester; m.p. l44.5-l47 C.; [011 185 (2 percent in dimethylformamide).

A solution of 280 g. of sodium hydroxide in 3.3 liters of water is added to a solution of 2.53 kg. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine methyl ester in 32.8 liters of methanol at 30 C. The mixture is stirred at 2025 C. for 4 hours and then diluted with 32.8 liters of water and filtered. The filtrate is treated with 3.65 liters of 2N hydrochloric acid and stirred for 16 hours. The insoluble product is collected on a filter, washed with water and dried. It is N-carbobenzoxy-D- phenylalanyl-D-phenylalanine; m.p. l55.5-157.5 C. following crystallization from acetone-petroleum ether; [01],, l2.7 (2 percent in chloroform).

EXAMPLE Salts of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine are prepared as follows.

With stirring, 1,000 g. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine is added to 4 liters of warm deionized water containing 2.20 ml. of 1,000N sodium hydroxide. The clear solution is frozen and lyophylized to give the product as a light-weight, fluffy white solid. It is N-carbobenzoxy-D-phenylalanyl-D-phenylalanine sodium salt; m.p. above 250 C. The product shows ultraviolet absorption maxima at 242, 248, 252, 258,

264, and 268 millirnicrons in absolute methanol.

With stirring, 1,000 g. of N-carbobenzoxy-D-phenylalanyl-D-phenylalanine is added to 4 liters of warm deionized water containing 2.20 ml. of 1,000N potassium hydroxide. The clear solution is frozen and lyophylized to give the product as a white solid. It is N-carbobenzoxy-D-phenylalanyl-D-phenylalanine potassium salt; m.p. above 250 C. The product shows ultraviolet absorption maxima at 242, 248, 252, 258, 264, and 268 millirnicrons in absolute methanol.

A solution is prepared by stirring 0.135 g. of 2- hydroxyethylamine and 1,000 g. of N-carbobenzoxy-D- phenylalanyl-D-phenylalanine in 4 liters of warm deionized water. The clear solution is frozen and lyophylized to give the product as a high melting white solid. It is N-carbobenzoxy-D-phenylalanyl-D-phenylalanine 2-hydroxyethylammonium salt.

I claim:

1. A pharmaceutical composition in the form of a tablet, capsule, lozenge, or pill possessing antiviral activity against measles virus and suitable for internal administration, com risin a harmac utical carrier and 25 to 1,000 mg. Ber dc sagg unit of a member of the class consisting of N-carbobenzoxy-D-phenylalanyl-D- phenylalanine and pharrnaceutically-acceptable salts thereof.

2. A composition according to claim 1 suitable for oral administration.

3. A method for the prophylaxis or treatment of measles infections in a mammal which comprises administering to said mammal a member of the class con- 

2. A composition according to claim 1 suitable for oral administration.
 3. A method for the prophylaxis or treatment of measles infections in a mammal which comprises administering to said mammal a member of the class consisting of N-carbobenzoxy-D-phenyl-alanyl-D-phenylalanine and pharmaceutically-acceptable salts thereof in an effective dose of at least 50 mg. per day.
 4. A method according to claim 3 wherein N-carbobenzoxy-D-phenylalanyl-D-phenylalanine is administered orally in an effective dose of at least 250 mg. per day for the prophylaxis or treatment of measles.
 5. A method according to claim 3 wherein N-carbobenzoxy-D-phenylalanyl-D-phenylalanine is administered parenterally for the prophylaxis or treatment of measles. 